The Lck tyrosine kinase molecule (p56.sup.lck), a lymphocyte specific, non-receptor type src-family protein kinase, plays a key role during the activation and early differentiation of T cells. This role has been described in the literature as follows. Defective TCR-mediated signaling observed in a lck mutant human Jurkat cell line was restored upon transfection of the cell with an intact gene (Straus and Weiss, 1992 Cell, 70, 585-593), suggesting a requirement for lck molecule in TCR-mediated signaling. Lck-deficient mice showed a dramatic reduction in double-positive (CD4.sup.+ CD8.sup.+) thymocytes, and no detectable single-positive thymocytes (Molina et al., 1992 Nature, 357, 161-164). Over-expression of a catalytically inactive version of Lck resulted in the maturational arrest of T cells at the G.sub.0 /G.sub.1 thymoblast stage (Levin et al., 1993 EMBO J., 12, 1671-1680).
Lck has been shown to associate physically and functionally with several molecules, such as CD4 and CD8 in the TCR complex (Shaw et al., 1989, Cell, 59, 627-636) and the interleukin-2 receptor .beta. chain (Hatakeyama et al., 1991 Science, 252, 1523-1528). The function of Lck is modulated by C-terminal Src kinase (CSK; Bergman et al., 1992 EMBO J., 11, 2919-2924; Chow et al., 1993 Nature, 365, 156-160; Autero et al., 1994 Mol. Cell. Biol., 14, 1308-1321) and tyrosine phosphatase CD45 (Mustelin and Altman, 1990 Oncogene, 5, 809-813; Mustelin et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 6302-6306; Ostergaard et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 8959-8963; Autero et al., 1994 Mol. Cell. Biol., 14, 1308-1321; reviewed by Mustelin and Burn, 1993 Trends Biochem. Sci., 18, 215-220).
Like other src-family protein kinases, Lck consists of three domains: the kinase, the Src homology region 2 ("SH2") and the Src homology region 3 ("SH3") domains. The SH2 domain of Lck has been reported to bind to tyrosine phosphorylated CD45 (Autero et al., 1994) and to ZAP-70 (Duplay et al., 1994 J. Exp. Med., 179, 1163-1172). ZAP-70 deficiency has been correlated with immunodeficiency in patients (Arpaia et al., Cell 76, 947-958, 1994). Since ZAP-70 binds to Lck and ZAP-70 deficiency is related to immunodeficiency, Lck binding is predictive of immunomodulatory activity for a binding protein.
The SH3 domain of Lck has been shown to associate with phosphatidylinositol (PI) 3-kinase (Prasad et al., 1993 Mol. Cell. Biol., 13, 7708-7717; Vogel and Fujita, 1993 Mol. Cell. Biol., 13, 7408-7417) and with p120 (Reedquist et al., 1994 Proc. Natl. Acad. Sci. USA, 91, 4135-4139); it is reported to participate in signal transduction or in membrane-cytoskelton interactions. Previous reports showed that the SH3 domain mediates protein-protein interactions via binding to proline-rich regions, such as those in 3BP-1,2 (Ren et al., 1993 Science, 259, 1157-1161), Sos (Li et al., 1993 Nature, 363, 85-88; Rozakis-Adcock et al., 1993 Nature, 363, 83-85), and Abl (Ren et al., 1994 Genes & Develop., 8, 783-795). Deletion or mutation of the SH3 domain usually activates the transforming potential of non-receptor type tyrosine kinases, suggesting that the SH3 domain negatively regulates the transforming activities of such proteins (Jackson and Baltimore, 1989 EMBO J., 8, 449-456; Hirai and Vermus 1990 Mol. Cell. Biol., 10, 1307-1318; Kato et al., 1986 Mol. Cell. Biol., 6, 4155-4160; Potts et al., 1998 Oncogene Res., 3, 343-355; Franz et al., 1989 EMBO J., 8, 137-147; Seidel-Dugan et al., 1992 Mol. Cell. Biol., 12, 1835-1845).
Proteins that directly associate with the SH3 domain in the c-abl proto-oncogene have been identified by the West-Western method (Cicchetti et al., 1992 Science, 257, 803-806), a method that allows direct isolation of genes encoding proteins that associate with target species such as the SH3 proteins. This procedure involves induction of proteins from a .lambda.gt 11 cDNA expression library and screening the proteins on nitrocellulose membranes using a protein or peptide probe. Previously with this method, several proteins have been identified that associate with intracellular signaling molecules or transcriptional factors, such as Max associating with Myc (Blackwood and Hisenman, 1991 Science, 251, 1211-1217), and 3BP-1 with the SH3 region of Abl (Cicchetti et al., 1992). These methods were employed in the present invention.
Alteration, preferably reduction, of the interaction of protein-tyrosine kinases and T-cell receptors (preferably CD4 or CD8) at the p56-lck binding site has been recognized for utility in the treatment of cancer, autoimmune disorders (e.g., SLE, multiple sclerosis, juvenile diabetes and rheumatoid arthritis), and infection with HIV and other viruses, for example as described in U.S. Pat. No. 5,250,431 with reference to certain modified T-cell CD4 and/or CD8 surface antigens.
Given the role of Lck in T cell activation and differentiation, it has been sought to identify and isolate gene(s) and protein(s) that associate with and modulate Lck, particularly the SH3 region of Lck. This has been accomplished in the present invention through the identification of LckBP1, including isolation of the murine LckBP1 gene, its incorporation as cDNA in a plasmid, and the expression of LckBP1 protein.